In humans, a vast range of psychiatric disease reflects disorder in neural circuits originating from prefrontal cortex (PFC). This is consistent with our understanding that PFC plays many functional roles in cognition, which include attention, inhibitory control, habit formation, and behavioral flexibility (Salzman and Fusi, 2010; Szczepanski and Knight, 2014). How does this relatively small brain region control such a diverse array of behavior? How is information flow into and out of PFC organized anatomically, not in terms of entire regions, but through targetable, molecularly identifiable, cell types? As an entry into this large problem, I will focus on ventromedial PFC (vmPFC), which is distinguished from other cortical regions by its extensive connectivity with the limbic system. vmPFC targets are key effectors of autonomic, emotional, and motivational behaviors, and its circuitry is linked to diseases of inflexible behavior such as addiction, obsessive-compulsive disorder, and post-traumatic stress disorder (Peters et al., 2009). Thus, a focused molecular interrogation of how the Layer V neurons forming the major outputs of this region are organized builds a necessary foundation to target complex psychiatric diseases with cell type specificity. The objective of this project is to determine how circuits initiated from Layer V projection neurons of vmPFC are organized at molecular and cellular resolution in the mouse. In Aim 1, I will use single-cell RNA sequencing to determine the molecular heterogeneity of these neurons, and then use retrograde labeling to determine the relation between their molecular identities and projection patterns. In Aim 2, I will take advantage of novel rabies-tracing (cTRIO) and iDISCO+ brain clearing/image registration tools to determine the anatomical organization of this circuit, focusing on how specific inputs are segregated towards specific outputs. Completion of this proposal will enable my future studies to test how PFC wiring specificity is generated, and how molecularly defined PFC neurons are recruited to specific behaviors that access cognitive function. For the field, this work will provide fundamental insight into the circuit architecture and molecular heterogeneity of PFC circuits. New markers and tools for genetic access to PFC subtypes will enable functional manipulation and therapeutic targeting of homologous circuits in humans. I will pursue these aims under the outstanding intellectual and technical guidance of my mentor Dr. Liqun Luo and collaborator Dr. Stephen Quake at Stanford, who are pioneers of the approaches used in my proposal. My career trajectory and research will further be guided by my advisory committee of Drs. Bill Newsome and Sue McConnell. The protected time of this K01 will allow me to fully take advantage of the training environment I have built over the last two years at Stanford. All of this directly facilitates my goal to lead a research program focused on how features of neural development and its resultant circuitry underlie cognition and behavior.